In the current proposal we have outlined a series of novel correlative studies for a national Phase II trial of interleukin-12 (IL-12) plus interferon-alpha (IFN-a) in patients with metastatic malignant melanoma (CALGB 500001). This trial is based upon a recently concluded NCI-sponsored phase I trial of IL-12 plus IFN-a in patients with advanced malignancy conducted at the Ohio State University (NCI T98-0020). IFN-a regulates gene expression in immune effectors and tumor cells via activation of the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway of signal transduction. We have previously shown that the cellular response to low-dose IFN-a can be markedly enhanced by pre-treating cells with IFN-gamma, a process that upregulates JAK-STAT protein levels. We subsequently confirmed the ability of IL-12 pre-treatments to sensitize patients to the effects of IFN-a in the phase I trial via a series of novel correlative studies utilizing patient serum, PBMCs, and tumor tissue. In the phase II trial, IL-12 will be administered at a dose of 300 ng/kg via the intravenous route on day 1. IFN-a2B will be administered subcutaneously at a dose of 3.0 x 10(6) U on days 2-6 following the dose of IL-12. This cycle of therapy will be repeated every other week. Overall response rate and time to disease progression will be the primary clinical endpoints of this trial. We hypothesize that this treatment regimen will result in the endogenous production of IFN-g, which in turn will upregulate levels of JAK-STAT signaling intermediates in patient PBMCs and tumor cells. We further hypothesize that upregulation of JAK-STAT signaling intermediates in patient PBMCs and tumor cells will lead to increased sensitivity of these tissues to low-dose IFN-a and increased expression of genes regulated by IFN-a. In Aim 1 we will measure serum levels of IFN-gamma and other immunomodulatory factors by ELISA. Real-Time RT PCR will be used to quantitate these factors at the transcript level. In Aim 2 we will measure levels of JAK-STAT signaling intermediates in patient PBMCs via intracellular flow cytometry, a technique that provides quantitative information utilizing very low numbers of cells. Also, tumor biopsies will be performed following the second dose of IL-12 and expression of JAK-STAT proteins will be analyzed via immunohistochemistry. In Aim 3, we will examine interferon-alpha-induced STAT signaling in patient PBMCs via flow cytometry utilizing an antibody specific for the phosphorylated form of STAT1. In addition, our laboratory now has the capacity to evaluate IFN-alpha-induced STAT activation within specific PBMC subsets using dual color flow cytometry. These techniques will provide us with a measure of the patient's sensitivity to IFN-a at the level of signal transduction. Finally, in Aim 4 we will determine the expression of IFN-regulated genes in patient PBMCs and tumor tissues in an attempt to quantitate the effects of this cytokine regimen on gene regulation. This series of experiments will permit us to evaluate the patient response to cytokine therapy at the cellular and molecular levels and identify key determinants of cytokine responsiveness.